Field of the Invention
The invention generally relates to the field of immunology.
Background Art
The natural immune system strikes a complex balance between highly aggressive, protective immune responses to foreign pathogens and the need to maintain tolerance to normal tissues. In recent years there has been increasing recognition that interactions among many different cell types contribute to maintaining this balance. Such interactions can, for example, result in polarized responses with either production of pro-inflammatory cytokines (e.g., interferon-gamma) by TH1 type T cells or production of interleukin-4 (IL-4) by TH2 type T cells that suppress TH1 activity. In a number of different animal models, T cell polarization to TH1 has been shown to favor protective immunity to tumors or infectious pathogens whereas T cell polarization to TH2 can be a critical factor in preventing development of cell-mediated autoimmune disease. The conditions that determine whether immune stimulation will result in aggressive cell-mediated immunity or in down regulation of such responses are highly localized in the sense that each tissue is comprised of a distinctive set of antigen presenting cells (APC) and lymphocyte lineages that interact to favor different immune responses. For example, under optimal conditions, the dendritic cells (DC) localized in a normal tissue may represent predominantly a lineage and stage of maturation that favors tolerogenic interactions and serves as a barrier to cell-mediated autoimmunity whereas a tumor or site of infection will attract mature myeloid dendritic cells that stimulate potent cell-mediated immune responses.
CD1d-restricted NKT cells are a unique class of non-conventional T cells that appear to play an important role in defining the outcome of immune stimulation in the local environment. They share with the larger class of NKT cells the expression of markers of both the T cell and natural killer (NK) cell lineages. As such, NKT cells are considered as part of innate immunity like NK cells and in humans their frequency in normal individuals can be as high as 2.0% of total T lymphocytes (Gumperz et al., 2002. J Exp Med 195:625; Lee et al., 2002. J Exp Med 195:637).
CD1d-restricted NKT cells are distinguished from other NKT cells by their specificity for lipid and glycolipid antigens presented by the monomorphic MHC class Ib molecule, CD1d (Kawano et al., Science 278 (1997), pp. 1626-1629). CD1d is a non-MHC encoded molecule that associates with β2-microglobulin and is structurally related to classical MHC class I molecules. CD1d has a hydrophobic antigen-binding pocket that is specialized for binding the hydrocarbon chains of lipid tails or hydrophobic peptides (Zeng et al., Science 277 (1997), pp. 339-345). CD1d is known to bind a marine sponge derived α-glycosylated sphingolipid, α-galactosylceramide (α-GalCer), and related molecules such as ceramide-like glycolipid antigens with α-linked galactose or glucose but not mannose (Kawano et al., Science 278 (1997), pp. 1626-1629; and Zeng et al., Science 277 (1997), pp. 339-345). As discussed below, the ability to activate CD1d-restricted NKT cells by stimulation with α-GalCer or related molecules bound to CD1d of antigen presenting cells has greatly facilitated functional analysis of this non-conventional T cell subset. In the absence of inflammation, CD1d-restricted NKT cells have been shown to localize preferentially in certain tissues like thymus, liver and bone marrow (Wilson et al., 2002. Trends Mol Med 8:225) and antitumor activity of NKT cells has been mainly investigated in mouse liver metastasis.
NKT cells have an unusual ability of secreting both TH1 and TH2 cytokines and potent cytotoxic as well as regulatory functions have been documented in inflammation, autoimmunity and tumor immunity (Bendelac et al., (1995) Science 268:863; Chen and Paul. 1997. J Immunol 159:2240; and Exley et al., 1997. J Exp Med 186:109).
Among the CD1d-restricted NKT cells is a subset, referred to herein as “iNKT cells,” that express a highly conserved αβT cell receptor (TCR). In man this invariant TCR is comprised of Vα24Jα15 in association with Vβ11 whereas in mice the receptor comprises the highly homologous Vα14Jα18 and Vβ8.2. Other CD1d-restricted NKT cells express more variable TCR. Both TCR invariant and TCR variant classes of CD1d-restricted T cells can be detected by binding of CD1d-tetramers loaded with α-GalCer (Benlagha et al., J Exp Med 191 (2000), pp. 1895-1903; Matsuda et al., J Exp Med 192 (2000), pp. 741-754; and Karadimitris et al., Proc Natl Acad Sci USA 98 (2001), pp. 3294-3298). CD1d-restricted NKT cells, as defined in this application (CD1d-restricted NKT), include cells that express either invariant or variant TCR and that bind or are activated by CD1d loaded with either α-GalCer or with related ceramide-like glycolipid antigens. CD1d-restricted NKT cells, as defined in this application (CD1d-NKT), include cells that express either invariant or variant TCR and that bind or are activated by CD1d loaded with either α-GalCer or with related sphingolipids that have α-linked galactose or glucose including molecules such as OCH, which differs from α-GalCer by having a shortened long-chain sphingosine base (C5 vs. C14) and acyl chain (C24 vs. C26) (Miyamoto et al., Nature 2001 413:531-4).
CD1d-restricted NKT have been shown to have direct cytotoxic activity against targets that express CD1d. It is likely, however, that the effect of CD1d-restricted NKT on immune responses is amplified through recruitment of other lymphocytes either by direct interaction or, perhaps even more importantly, by indirect recruitment through interaction with DC. CD1d-restricted NKT have the unique ability to secrete large quantities of IL-4 and IFN-γ early in an immune response. Secretion of IFN-γ induces activation of DC which produce interleukin-12 (IL-12). IL-12 stimulates further IFN-γ secretion by NKT cells and also leads to activation of NK cells which secrete more IFN-γ.
Since CD1d-restricted NKT are able to rapidly secrete large amounts of both IL-4 and IFN-γ, the polarization of immune responses will depend on whether the effect of pro-inflammatory IFN-γ or anti-inflammatory IL-4 cytokines predominate. This has been reported to be, in part, a function of the relative frequency of different subsets of CD1d-restricted NKT. These subsets include (i) an invariant CD1d-restricted NKT population that is negative for both CD4 and CD8 and that gives rise to predominantly a TH1 type response including secretion of pro-inflammatory IFN-γ and TNF-α and (ii) a separate population of CD1d-restricted NKT that is CD4+ and that gives rise to both a TH1 type and TH2 type response including secretion of the anti-inflammatory Th2-type cytokines IL-4, IL-5, IL-10 and IL-13 (Lee et al., J Exp Med 2002; 195:637-41; and Gumperz et al., J Exp Med 2002; 195:625-36). In addition, NKT cell activity is differentially modulated by depending on the particular ceramide-like glycolipid bound to CD1d (see, e.g., US Patent Application Publication No. 2006/0052316). Local factors that influence activation of CD1d-restricted NKT subsets include the cytokine environment and, importantly, the DC that are recruited to that environment.
A number of indirect mechanisms contribute to the protective effect of CD1d-restricted NKT cells. Activation of NKT cells by administration of α-GalCer in vivo results in concomitant activation of NK cells (Eberl and MacDonald, Eur. J. Immunol. 30 (2000), pp. 985-992; and Carnaud et al., J. Immunol. 163 (1999), pp. 4647-4650). In mice deficient in NKT cells, α-GalCer is unable to induce cytotoxic activity by NK cells. NKT cells also enhance the induction of classical MHC class I restricted cytotoxic T cells (Nishimura et al., Int Immunol 2000; 12:987-94; and Stober et al., J immunol 2003; 170:2540-8).
The availability of a defined antigen, e.g., α-GalCer and related antigens, that can be employed to specifically activate CD1d-restricted NKT cells has made it possible to examine the role of these non-conventional T cells in a variety of immune responses.
Indeed, α-GalCer has significant promise as a therapeutic agent or adjuvant. For example, α-GalCer administration has a dramatic effect on a number of different microbial infections, including protective effects in murine malaria, fungal and hepatitis B virus infections (Kakimi et al, J Exp Med 192 (2000), pp. 921-930; Gonzalez-Aseguinolaza et al., Proc Natl Acad Sci USA 97 (2000), pp. 8461-8466; and Kawakami et al., Infect Immun 69 (2001), pp. 213-220). Dramatic effects of administration of α-GalCer have also been observed in animal models of tumor immunity. For example, stimulation with α-GalCer suppresses lung and liver metastases in an NKT dependent manner (Smyth et al., 2002. Blood 99:1259). In addition, α-GalCer has been shown to have a protective effect against certain autoimmune diseases, including type 1 diabetes a,d experimental autoimmune encephalomyelitis (EAE, a well-known model system for multiple sclerosis) (Hong S, et al. Nat. Med. 2001; 7:1052-1056 and Miyamoto K. et al. Nature. 2001; 413:531-534).
However, NKT cells, upon restimulation with α-GalCer, become unresponsive, e.g., reduced in their capacity to proliferate, produce cytokines, transactivate other cell types, and prevent tumor metastasis. Parekh, V V, et al. J. Clin. Invest. 115:2572-2583 (2005). Accordingly, there remains a 0need in the art for methods of stimulating NKT cells multiple times without causing the NKT cells to become nonresponsive.